Mem Inst Oswaldo Cruz, Rio de Janeiro, 102(1) February 2007
Phlebotomine sand flies (Diptera: Psychodidae) associated with changing patterns in the transmission of the human cutaneous leishmaniasis in French Guiana
ICellule d'Intervention Biologique d'Urgence, Institut Pasteur, 25, rue du Dr. Roux, 75724 Paris cedex 15, France
IILaboratoire d'Entomologie médicale, Institut Pasteur de la Guyane, Cayenne cedex, French Guiana
IIILaboratoire de Parasitologie Mycologie, Faculté de Pharmacie, Châtenay-Malabry, France
IVLaboratoire de Parasitologie, Centre National de Référence des Leishmania, Montpellier, France
Between March 2000 and December 2001 a survey of the sand flies (Diptera: Phlebotominae) of French Guiana was carried out during 14 nights of captures with CDC light-traps and Malaise traps, and resulted in the collection of 2245 individuals of 38 species. The most abundant species were Lutzomyia (Trichophoromyia) ininii Floch & Abonnenc, Lu.(Psychodopygus) squamiventris maripaensis Floch & Abonnenc, and Lu .(Nyssomyia) flaviscutellata Mangabeira. Half of the collected sand flies females were dissected under field conditions and five species were found harboring Leishmania-like parasites. The Leishmania (Kinetoplastidae: Trypanosomatidae) species were identified by molecular typing, and for the first time Lu. (Nys.) flaviscutellata was found harboring Leishmania (Viannia) guyanensis and Lu. (Tri) ininii harboring unknown Leishmania. The first record for French Guiana of Lu. (Psy.) squamiventris maripaensis harboring L. (V.) naiffi, was also reported. The patterns of diversification of the human cutaneous leishmaniasis transmission in French Guiana are discussed.
With about 300 cases of human cutaneous leishmaniasis (HCL) reported annually (Carme 2001, Lightburn et al. 2001) and some extreme situations (Couppié et al. 2004), this disease still represents a major public health problem in French Guiana. The first HCL cases were confirmed in French Guiana in 1943 (Floch 1943) and past studies showed that Leishmania (Viannia) guyanensis is the predominant species of Leishmania (Floch 1957, Dedet et al. 1989, Raccurt 1996) but L. (L.) amazonensis, L. (V.) braziliensis, L. (V.) lainsoni, and L. (V.) naiffi have also been diagnosed (Dedet et al. 1985, Raccurt et al. 1995, Basset et al. 2001, Pratlong et al. 2002). The sand flies of French Guiana have been extensively studied until the 1990s and 77 sand flies species were reported (Floch & Abonnenc 1952, Léger et al. 1977). The entomological studies have shown that the sand fly Lutzomyia (Nyssomyia) umbratilis Ward & Fraiha was the major vector of L. (V.) guyanensis (Le Pont & Pajot 1980, Pajot et al. 1982). The other sand flies species found infected with Leishmania parasites were Lu. (Nys.) flaviscutellata Mangabeira, harboring L. (L.) amazonensis (Dedet et al. 1989) and Lu. (Lut.) gomezi Nitzulescu and Lu. (Psy.) yullii pajoti Abonnenc, Léger & Fauran harboring unidentified Leishmania species (Killick-Kendrick 1990, Young & Duncan 1994). The sand flies species transmitting L. (V.) braziliensis, L. (V.) lainsoni, and L. (V.) naiffi are partly known in other South-American countries (Naiff et al. 1991, Young & Duncan 1994, Feliciangeli 2004), but have not been so far identified in French Guiana. Furthermore, in the last ten years, the geographical distribution of the HCL transmission in French Guiana has changed, previous foci have disappeared and new foci are reported. The people at risk for HCL contamination have also changed and now the military personnel represent 25 to 85% of the annual number of cases (Lightburn et al. 2001). Finally, the environment of French Guiana is continuously changing due to human pressure on the forested area for road construction, housing development, and gold mining.
For these reasons, a research program to update our knowledge on the HCL transmission in French Guiana was started in 2000. The objectives of the program were to identify the species of sand flies acting as vector of HCL in the probable contamination places, to identify the Leishmania parasites when present in the collected sand flies, to characterize the foci and finally to collect information on the biology and ecology of the species.
MATERIALS AND METHODS
The study sites - From March 2000 to December 2001, 14 collections of sand flies were carried out in different areas of French Guiana. The prospected locations were chosen in relation with the report of HCL cases during the past weeks or the month before the captures. The report of the HCL cases remained anonymous and only geographical locations were given by the Direction Départementale des Affaires Sanitaires et Sociales (DDASS) reports and the military headquarters for the study. The procedures of the report of human cases were in accordance with the ethical standards and the research program was approved by the DDASS of French Guiana representing the French Ministry of Health.
Different ecological conditions of French Guiana were prospected for sand flies collection at different periods of the year. The forested areas along the littoral at Regina were prospected in March, April, October, and November 2000, and at Nancibo in December 2001 (Fig. 1). The forested areas in the interior of the country at Maripasoula and the Upper Maroni River were prospected in October 2000 and at Saül in December 2000. The savannas areas at Montsinery were prospected in February and March 2001 and at Sinnamary in September 2001. Finally, some agricultural areas were also prospected at Cacao in June 2001, Montsinery in July 2001 and Saül in October 2001 (Fig.1, Table).
The sand flies collections and species identification - Each collection of sand flies included two consecutive nights of capture with three types of traps, one CDC light-trap (Bioquip model), one modified CDC light-trap (with a green plastic hat and a more powerful light-bulb, Gantier model) and one Malaise trap. One or two traps of each type were left between 6 p.m. and 7 a.m. in different places (close to trees, bushes, armadillos' hole, etc.) and at different heights, varying from 0.30 to 10 m. The sand flies were collected in the traps at regular intervals during the nights. The intervals were in most cases, each 3 h, which is at 9 p.m., 12 p.m., 3 a.m., and 6 a.m.
The collected sand fly females were dissected under field conditions just after the empting of the trap, to search for natural infection to Leishmania spp. The infected sand flies were kept in Dulbecco's Modified Eagle's Medium (ref D5523, Sigma Chemicals Co.) supplemented with fetal bovine serum (ref F2442, Sigma) and brought to the Laboratory of Immunology of the Pasteur Institute of French Guiana for a tentative culture of the Leishmania parasite. Some of the positive samples were also sent to the Laboratory of Parasitology of the Faculty of Medicine of Montpellier for identification by molecular typing, which was carried out by systematic sequencing of a part of the RNA pol II large sub-unit gene.
All sand flies collected were brought to the Laboratory of Entomology and processed for identification. Processing and mounting of the sand flies males and females was made according to the following protocol (Tran-Kiem 2000): the individuals were rinsed in distilled water until flotation, then left 10 min in the warmed up Marc-André Liquid (distilled water 300 g, chloral hydrate 400 g, acetic acid 300 g) for lightening, then rinsed twice with alcohol 90°, then mounted in a drop of Creosote, the exceeding of Creosote was removed and the Euparal mounting liquid was added. The species were then identified firstly with the informatics' key (Lebbe et al. 1987) and confirmed with the available descriptions (Floch & Abonnenc 1952, Young & Duncan 1994). The Laboratory of Mycology of the Faculty of Pharmacy of Châtenay-Malabry (Paris IX) further confirmed the species identifications. The molecular confirmation of the Lutzomyia species was not performed due to the absence of reliable methods for the sand flies species of French Guiana.
A total of 14 collections were carried out and 2245 sand flies were collected including 1265 females, and 980 males, among which 1865 individuals (83%) were identified (1100 females and 765 males) (Table). A lot of individuals (17%) could not be identified because they were either damaged during the transportation between the field and the laboratory, damaged during the laboratory process of lightening and mounting, or because the specimens could not be attributed to a species description. A total of 38 species were collected, which is close to the 43 species reported during the last sand flies survey in French Guiana (Léger et al. 1977), and about half of the 78 species previously described from French Guiana (Floch & Abonnenc 1952, Oliveira et al. 2001). The most abundant species in our survey were Lu. (Tri.) ininii, Lu. (Psy.) squamiventris maripaensis and Lu. (Nys.) flaviscutellata and the 3 species were found harboring Leishmania-like flagellates parasites (Table). The most abundant species reported during the 1977 survey were Lu. (Tri.) ubiquitalis Mangabeira followed by Lu. (Tri.) ininii, and Lu. (Psy.) squamiventris maripaensis. Thus, the overall results do not show any significant changes in the species diversity and densities, except for the high density of Lu. (Tri.) ubiquitalis collected in Maripasoula in 1977 (Léger et al. 1977). The collection made in Maripasoula during our survey resulted in 32 individuals that could not be identified.
About half (534 females, i.e. 48.5%) of the sand flies females collected were dissected in the field, examined from the presence of Leishmania parasites and placed on culture when found positive. Five sand flies females were found harboring Leishmania-like flagellates parasites, with an overall infection rate of 0.84%. Four positive sand flies could be identified. No Leishmania culture was successful, but molecular typing on four of these Leishmania field samples allowed the identification of 2 Leishmania species. One female of Lu. (Nys.) flaviscutellata was found harboring L. (V.) guyanensis and one female of Lu. (Psy.) squamiventris maripaensis was found harboring L. (V.) naiffi. The other two molecular typing were significant of a Leishmania genus, but the species were not identified.
The highest sand flies density and species diversity were found during high and medium rainfalls periods, in natural and forested areas (December 2001 at Nancibo). In the contrary, the lowest sand flies density were found during the dry season, in forested and savanna areas, submitted to important ecological changes due to military activities (Regina in August and October 2000), agricultural practices (Sinnamary in September 2001), and gold mining activities (Saül in December 2000) (Fig. 2). The infected sand flies females were collected in October and November 2000 at Regina, in February and July 2001 at Montsinery and in September 2001 at Sinnamary.
This is the first report of Lu. (Nys.) flaviscutellata harboring L. (V.) guyanensis. This species is also considered a proven vector of L. (L.) amazonensis (Killick-Kendrick 1990). This positive sand fly was collected in Regina in November 2000, and the overall infection rate for this species was 2.32%. During the same capture, only one female of Lu. (Nys.) umbratilis, the proven vector of L. (V.) guyanensis in French Guiana (Le Pont & Pajot 1980) was collected (Table). The second species of Leishmania identified, L. (V.) naiffi was found in a female of Lu. (Psy.) squamiventris maripaensis. This sand fly species was reported as a vector of L. (V.) naiffi in Brazil (Naiff et al. 1991), but this is the first time that Lu. (Psy.) squamiventris maripaensis can be considered a vector of L. (V.) naiffi in French Guiana, where the HCL species has already been reported in human cases (Basset et al. 2001, Pratlong et al. 2002). The positive female of Lu. (Psy.) squamiventris maripaensis was collected in September 2001 in the savanna area of Sinnamary and the overall infection rate for this species was 1.78%. Two females of Lu. (Tri.) ininii were found harboring unidentified Leishmania flagellates with an overall infection rate of 1.47%, and this could be the first report of Lu. (Tri.) ininii as probable vector of HCL.
The results are showing a natural Leishmania circulation in French Guiana expanding all year long and widespread in the country, in particular in areas submitted to important ecological changes due to military activities (Regina) and agricultural practices (Montsinery and Sinnamary). The infected sand flies were collected during periods with low rainfalls (October 2000 and September 2001) and medium rainfalls (November 2000 and February 2001). However, the highest infection rates were found in September 2001 at Sinnamary (0.33) and November 2000 at Regina (0.14). Furthermore, when the densities of the potential vectors are observed, Lu. (Nys.) flaviscutellata was most abundant in November 2000 (at Regina) and December 2001 (at Nancibo), Lu. (Tri.) ininii was must abundant in November 2000 (at Regina) and Lu. (Psy.) squamiventris maripaensis was most abundant in December 2001 (at Nancibo), confirming a transmission season mostly at the end of the year when the dry season is also ending and the rainfalls start to increase (Fig. 2). Some HCL foci could be detected at Regina, Montsinery and Sinnamary, with not only report of clinical cases but also presence of infected sand flies.
Although, the annual HCL incidence do not show a recent increase in French Guiana because 2.3 cases per 1000 inhabitant were reported in 1989 (Dedet 1990) against 1.5 to 2 cases per 1000 inhabitant reported between 1986 and 2000 (Carme et al. 2001), the transmission patterns are changing with an increase in the number of Leishmania species diagnosed (Basset et al. 2001) and the report of new sand flies vector. HCL is probably endemic in the Amazonian region since the arrival of the first human beings (Andrade Fiho & Brazil 2003) and this is demonstrated by the great diversity of HCL transmission found in this region. French Guiana is included in the Amazon geographic area and the small number of Leishmania parasites and sand flies vectors previously found in this country may be due to a report of cases mostly for the population living along the littoral area. Actually, French Guiana is submitted to an important human pressure of deforestation for gold mining, road construction and agricultural practices. Consequently, the population exposed to the natural leishmaniasis forested cycles is increasing and the HCL transmission is gaining in diversity. The first report of three sand flies species found infected with Leishmania parasites in French Guiana, Lu. (Nys.) flaviscutellata for L. (V.) guyanensis, Lu. (Psy.) squamiventris maripaensis for L. (V.) naiffi, and Lu. (Tri.) ininii for unknown Leishmania species is the major finding of this survey, and suggests that more extensive studies should be undertaken for a better knowledge of HCL transmission patterns and prevention in the Amazonian ecosystem of French Guiana.
To the technicians of the Centre National de Reference des Leishmania from CHU Montpellier for molecular typing of the Leishmania parasites. To the DASS-Guyane and the French Army Headquarters in French Guiana for providing information on the cases as well as their help in some restricted forested areas. To Dr Patrick Rabarison of the Laboratory of Medical Entomology of the Pasteur Institute of French Guiana, Dr Jean-Louis Sarthou, former Director of the Pasteur Institute of French Guiana for his support, Dr Jacques Morvan, Director of the Pasteur Institute of French Guiana, and the Laboratory of Immunology of the Pasteur Institute of French Guiana for the tentative cultures of Leishmania and the support in the work.
Andrade Filho JD, Brazil RP 2003. Relationships of New World phlebotomine sand flies (Diptera: Psychodidae) based on fossil evidence. Mem Inst Oswaldo Cruz 98: 149-154.
Basset D, Pratlong F, Ravel C, Puechberty J, Dereure J, Dedet JP 2001. Les leishmanioses déclarées en France en 1999. Bull Epid Hebd 5: 1-5.
Carme B 2001. Human parasitoses in French Guiana. Presse médicale 30: 1601-1608.
Carme B, Aznar C, Pradinaud R 2001. Absence of proven resurgence of Chagas disease or cutaneous leishmaniasis in French Guiana over the last two decades. Ann Trop Med Parasitol 95: 623-625.
Couppié P, Clyti E, Sainte-Marie D, Dedet JP, Carme B, Pradinaud R 2004. Disseminated cutaneous leishmaniasis due to Leishmania guyanensis: a case of a patient with 425 lesions. Am J Trop Med Hyg 71: 558-560.
Dedet JP 1990. Cutaneous leishmaniasis in French Guiana: a review. Am J Trop Med Hyg 43: 25-28.
Dedet JP, Pradinaud R, Desjeux P, Jacquet-Viallet P, Girardeau I, Esterre P, Gotz W 1985. The 2 first cases of cutaneous leishmaniasis due to Leishmania mexicana amazonensis in French Guiana. Bull Soc Path Exot 78: 64-70.
Dedet JP, Pradinaud R, Gay F 1989. Epidemiological aspects of human cutaneous leishmaniasis in French Guiana. Trans R Soc Trop Med Hyg 83: 616-620.
Feliciangeli MD 2004. Natural breeding places of phlebotomine sandflies. Med Vet Entomol 18: 71-80.
Floch H 1943. Rapport sur le fonctionnement technique de l'Institut Pasteur de la Guyane pendant l'année 1943. Pub Inst Pasteur Guyane française territ Inini 75: 1-79.
Floch H 1957. Epidemiology of American forest leishmaniasis in French Guiana. Riv Malariol 36: 233-242.
Floch H, Abonnenc E 1952. Diptères phlébotomes de la Guyane et des Antilles Françaises. Faune de l'Union Française 14: 1-207.
Killick-Kendrick R 1990. Phlebotomine vectors of the leishmaniasis: a review. Med Vet Entomol 4: 1-24.
Lebbe J, Vignes R, Dedet JP 1987. Computer Aided Identification of Phlebotomine Sandflies of French Guiana (Diptera: Psychodidae), Publication de l'Institut Pasteur de la Guyane française, Cayenne, 165 pp.
Léger N, Abonnenc E, Pajot FX, Kramer R, Claustre J 1977. Liste commentée des phlébotomes de la Guyane Française. Cah ORSTOM, sér Entomol méd Parasitolo XV: 217-232.
Le Pont F, Pajot FX 1980. La leishmaniose en Guyane française. Etude de l'écologie et du taux d'infection naturelle du vecteur Lutzomyia (Nyssomyia) umbratilis Ward & Fraiha 1977 en saison sèche. Considérations épidémiologiques. Cah ORSTOM, sér Entomol méd Parasitolo XVIII: 359-382.
Lightburn E, Meynard JB, Morand J, Garnotel E, Kraemer P, Hovette P, Banzet S, Dampierre H, Lepage J, Carme B, Pradinaud R, Morillon M, Dedet JP, Chouc C, Boutin JP 2001. Surveillance épidémiologique des leishmanioses tégumentaires en Guyane Synthèse de données militaires sur 10 ans. Méd Trop 62: 545-553.
Naiff RD, Freitas RA, Naiff MF, Arias JR, Barrette TV, Momen H, Grimaldi JG 1991. Epidemiological and nosological aspects of Leishmania naiffi Lainson & Shaw, 1989. Mem Inst Oswaldo Cruz 86: 317-321.
Oliveira AG, Andrade Filho JD, Falcão AL, Brazil RP 2001. A new sand fly, Lutzomyia campograndensis sp. n. (Diptera: Psychodidae: Phlebotominae) from the state of Mato Grosso do Sul, Brazil. Mem Inst Oswaldo Cruz 96: 325-329.
Pajot FX, Le Pont F, Gentile B, Besnard R 1982. Epidemiology of leishmaniasis in French Guiana. Trans R Soc Trop Med Hyg 76: 112-113.
Pratlong F, Deniau M, Darie H, Eichenlaub S, Pröli S, Garrabe E, Le Guyadec T, Dedet JP 2002. Human cutaneous leishmaniasis caused by Leishmania naiffi is widespread in South-America. Ann Trop Med Parasitol 96: 781-785.
Raccurt CP 1996. Cutaneous leishmaniasis and leishmania species in French Guiana. Méd Trop 56: 415.
Raccurt CP, Pratlong F, Moreau B, Pradinaud R, Dedet JP 1995. French Guiana must be recognized as an endemic area of Leishmania (Viannia) braziliensis in South America. Trans R Soc Trop Med Hyg 89: 372.
Tran-Kiem AT 2000. Contribution à l'Etude des Phlébotomes de la Guyane Française, Thesis, Faculté de Pharmacie de Chatenay-Malabry, Chatenay-Malabry, 312 pp.
Young DG, Duncan MA 1994. Guide to the identification and geographic distribution of Lutzomyia sand flies in Mexico, the West Indies, Central and South-America (Diptera: Psychodidae). Mem Am Entomol Instit 54: 1-881.